1. Field of the Invention
This invention relates to a class of proteoglycans having fucosylated acidic glycan side chains bound to a protein backbone which have been found to stimulate selectively proliferation of natural killer (NK) cells and/or xcex3xcex4T cells. They are useful as immunostimulants, e.g., in the treatment of cancer and viral infections.
2. Description of the Related Art
The proteoglycans of the invention are produced by proliferating cells, for example by sponge cells, sea urchin cells, and, in the case of higher animals (including humans), by embryonic cells and tumor cells. In the natural proteoglycan form, the compounds are large (ca. 5000 to 30,000 kD) extracellular or membrane-bound molecules having a protein backbone which is glycosylated with acidic glycan chains having an unusual polysaccharide sequence containing internal fucose. The structure of the acidic glycan side chains of the proteoglycan isolated from the marine sponge Microciona prolifera has been partially characterized (Spillmann, et al,, J. Biol. Chem. (1993) 268: 13378-13387, contents incorporated herein by reference), and we have previously shown that this proteoglycan is involved in cellular aggregation (Misevic, et al., J. Biol. Chem. (1987) 262: 5870-5877; Misevic, et al., J. Biol. Chem. (1990) 265: 20577-20584; Misevic, et al., J. Biol. Chem. (1993) 268: 4922-4929, contents of all of these articles incorporated herein by reference). The previously undescribed protein backbone of the Microciona prolifera proteoglycan has now been isolated and characterized, and novel proteoglycans derived from sponges of other genera have also been characterized, as described below.
Those concerned with these and other problems recognize the need for an improved fucose containing proteoglycan or acidic glycan and their pharmaceutical use.
It has now surprisingly been discovered that these compounds are potent stimulators of NK cells and xcex3xcex4T cells. It particular, compounds of the invention isolated from an organism of all the phyla and preferably:
from organisms of the Phylum Porifera e.g., of the class Demospongiac, especially of the order Poccilosclerida, family Microcionidae (e.g., of the genus Microciona), or family Mycalidae (e.g., of the genus Mycale), or the order Halichondrida, family Halichondridae (e.g., of the genus Halichondria), or the order Hadromerida, family Clionidae (e.g., of the genus Cliona), or the order Haplosclerida, family Haliclonidae (e.g., of the genus Haliclona),
and/or from organisms of the phylum Echinodermata.
These compounds have been shown to stimulate selectively different clones of NK cells and T cells. Moreover, it has been found that compounds of the invention have significant anticancer, especially antimetastatic, effects in vivo. It is believed that these anticancer effects are due to stimulation in vivo of NK cells and/or xcex3xcex4T cells. The precise mechanism of this stimulation is unclear, but without intending to be bound by a particular theory, we suggest that these cells may be stimulated by polyvalent interactions with fucosylated acidic glycans of the class described herein and in this way can identify and destroy hyperproliferating cells expressing similar glycan structures. In a pathogenic case, where the hyperproliferating cells are not destroyed in this manner, it is believed that although the hyperproliferating cells produce these acidic glycans, they shed them or present them in monovalent form or other nonstimulatory or inhibitory form, thereby evading detection and destruction by NK cells and/or xcex3xcex4T cells specific for such acidic glycans. Application of the compounds of the invention stimulates NK cells and/or xcex3xcex4T cells specific for such cancer cells, thereby leading to their destruction. Additionally, the compounds of the invention are useful for stimulating NK cells and/or xcex3xcex4T cells against viral or retroviral infections. Finally, in monovalent form, the compounds of the invention are useful for inhibiting the activation of NK cells and/or xcex3xcex4T cells, thereby finding utility as immunosuppressants.
The compounds of the invention are selective in their action, in that particular compounds of the invention stimulate only particular clones or subpopulations of NK cells or xcex3xcex4T cells. No significant stimulation of B cells or xcex3xcex4T cells is observed, so undesirable immunostimulation, e.g., an allergenic or autoimmune response, is avoided. Despite this selectivity, all humans tested, from a variety of ethnic and racial groups, have cell populations capable of being significantly stimulated by the compounds of the invention. Compounds having the glycan structures of the class described herein are found in a wide variety of hyperproliferating cells from sponges to human tumors, thus the basic structure of the compounds is highly conserved. It is hypothesized that compounds of the class described herein act as signals for stimulating the body""s defenses against unwanted proliferation of cancerous or infected cells, and that cancers or resistant viral infections may arise when, as described above, these compounds are secreted in nonstimulatory form. Among the examples described herein, it is noted that compounds of the invention isolated from those of the genus Microciona are more effective in stimulating NK cells, as described in example 1 below, whereas compounds isolated from the genus Halichondria are more effective in stimulating xcex3xcex4T cells, as described in example 9, thus selectivity among cell types receptive to this stimulation is also possible.
Other objects, advantages, and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.
The invention thus provides
1. Fucose-containing proteoglycans and acidic glycans, and/or fragments thereof, preferably proteoglycans, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of all the phyla and preferably from an organism of the phylum Porifera, e.g., as described above, especially of the genera Microciona and/or Halichondria and/or Mycale and/or Cliona and/or from an organism of the phylum Echinodermata especially of the genus Lytechinus for use as a pharmaceutical or therapeutic agent in vivo or for ex vivo therapy; and pharmaceutical compositions comprising such compounds in combination with a pharmaceutically acceptable carrier or diluent.
2. Novel fucose-containing proteoglycans and acidic glycans, and or fragments thereof, preferably proteoglycans, isolated or capable of being isolated from organisms of the genus Halichondria and/or Mycale and/or Cliona.
3. Novel fucose-containing acidic glycans capable of being isolated from a sea urchin of the genus Lytechinus,
4. A Fucose-containing acidic glycan for use as a pharmaceutical or therapeutic agent in vivo or for ex vivo therapy; and pharmaceutical compositions comprising such compounds in combination with a pharmaceutically acceptable carrier or diluent; and capable of binding to monoclonal antibodies of the type of these named xe2x80x9cBlock 2xe2x80x9d and described in the reference xe2x80x9cMisevic, et al., J. Biol. Chem. (1993) 268: 4922-4929,
5. A method of stimulating the proliferation of mammalian, e.g., human, NK cells and/or xcex3xcex4T cells comprising contacting said cells with a compound of the invention (a fucose-containing proteoglycan and acidic glycan, and/or fragment thereof, preferably a proteoglycan and/or fragment(s) thereof, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of the phylum Porifera, or Echinodermata e.g., as described above, especially of the genera Microciona and/or Halichondria and/or Mycale and/or Cliona, and/or of the phylum Echinodermata especially of the genus Lytechinus, in an ex vivo setting or in vivo, e.g., as a vaccine; or a method of treating cancer (e.g., preventing or inhibiting onset, growth, or metastasis of a tumor), or treating or preventing a viral or retroviral infection, in a mammal, e.g., man; comprising administering a pharmaceutically effective amount of a compound of the invention to a patient in need of such treatment; or the use of a compound of the invention in the manufacture of a medicament for treatment or prevention of cancer or viral or retroviral infections.
6. The use of a fucose-containing proteoglycan or acidic glycan, or fragment thereof, preferably a proteoglycan and/or fragment(s) thereof, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of the phylum Porifera and/or Echinodermata, e.g., as described above, especially of the genera Microciona and/or Halichondria, and/or Mycale and/or Cliona for the phylum Porifera, especially of the genus Lyiechinus for the phylum Echinodermata for ex vivo stimulation of proliferation of NK cells and/or xcex3xcex4T cells.
7. A method for screening for or detecting an immunosuppressive (e.g., NK cell and/or xcex3xcex4T cell) inhibitory compound comprising measuring proliferation of NK cells and/or xcex3xcex4T cells in a system containing an NK cell or xcex3xcex4T cell stimulatory concentration of a compound of the invention in the presence and absence of a test compound; and compounds identified using such a method.
8. A gene coding for a protein capable of post-translational glycosylation to form the proteoglycan of the invention, vectors containing such a gene, and transformed cells, especially (i) production cells, e.g., sponge cells, incorporating such a gene for use in producing the desired proteoglycan at enhanced levels or (ii) cancer cells removed from a patient, transformed with the gene so as to express the proteoglycan in stimulatory form, irradiated, and reintroduced into the patient. The gene for Microciona proteoglycan can be isolated, for example, using oligonucleotide probes of a cDNA library based on the disclosed amino acid sequences.
Appropriate dosages of the compounds of the invention will of course vary, e.g. depending on the condition to be treated (for example the disease type or the nature of resistance), the effect desired, and the mode of administration. In general however satisfactory results are obtained on administration orally, rectally, nasally, topically, or parenterally, e.g. intravenously, for example by i.v. drip or infusion, at dosages on the order of from 0.01 to 2.5 up to 5 mg/kg, e.g. on the order of from 0.05 or 0.1 up to 1.0 mg/kg. Suitable dosages for patients are thus on the order of from 0.5 to 125 up to 250 mg i.v., e.g. on the order of from 2.5 to 50 mg i.v. Pharmaceutical compositions of the invention may be manufactured in conventional manner, in a suitable aqueous carrier, for example sterile buffered physiological saline.
For ex vivo stimulation of cells, as described more fully in the example below, a suitable amount, e.g., at least 10 ml, of the patient""s blood is removed, peripheral blood mononuclear cells are isolated from the blood, placed in a complete medium in the presence of a stimulatory concentration of a compound of the invention, e.g., 10-500 xcexcg/ml, ca. 100 xcexcg/ml, optionally in the presence of IL-2, and the culture is maintained until a significant increase in the population of the desired cell type is observed; e.g., ca. 2-4 weeks. Following stimulation of the cells, the cells are isolated from the medium, placed in an injection solution, e.g., sterile buffered physiological saline or plasma, and injected back into the patient. The compound of the invention for this use can, for example, be a proteoglycan or acidic glycan derived from a marine sponge as described in the examples, but may also be a proteoglycan, acidic glycan or fragment thereof isolated from a culture of the cancerous cells to be treated.
The compounds can be useful notably as pharmaceuticals, particularly as immunostimulants, e.g. in the treatment of cancer and viral infections.